The present invention pertains to substrates for performing multiple assays of biological or chemical analytes. More particularly, the invention relates to a porous inorganic substrate for attaching an array of biological or chemical molecules and a method of fabricating such substrates.
Clinical and research laboratories are increasingly using DNA testing as a means to determine genetic risk factors for diseases like breast cancer, heart disease, Alzheimer""s disease, etc. Simultaneous screening for many risk factors is possible by printing many xe2x80x9cmicrodotsxe2x80x9d of DNA onto the same substrate, typically either a porous, organic membrane or a flat, non-porous glass slide to form a high-density array. High-density arrays have become useful tools for drug researchers and geneticists to obtain information on the expression of genes. A high-density array typically comprises between 2,000 and 50,000 probes in the form of single stranded DNA, each of a known and different sequence, arranged in a predetermined pattern on a substrate.
The arrays are used to test whether single stranded target DNA sequences interact or hybridize with any of the single stranded probes on the array. The testing procedure consists of printing and binding single-stranded DNA molecules onto a substrate. The substrate may be any size, but typically takes the form of a standard 1 inchxc3x973 inch microscope slide. The printed DNA sequence is for a known genetic risk factor and may be tagged with a fluorescent marker for identification. Unknown DNA, such as obtained from a patient, is tagged with a different fluorescent marker and washed over the slide for a specified period of time and then rinsed. If the unknown DNA contains any strands that have complementary nucleic acid sequences to the known strand, hybridization occurs. Any hybridization on the rinsed slide is detected as fluorescence from the marker on the unknown DNA. Fluorescence above a predetermined, threshold intensity indicates that the unknown DNA contains the genetic risk factor associated with the known DNA printed on the slide.
After exposing the array to target sequences under selected test conditions, scanning devices can examine each location on the array and determine the quantity of targets that are bond to complementary probes. The ratio of fluorescent intensity relative to a reference at each spot on the high-density array provides the relative differential expression for a particular gene. DNA arrays can be used to study the regulatory activity of genes, wherein certain genes are turned on or xe2x80x9cup-regulatedxe2x80x9d and other genes are turned off or xe2x80x9cdown-regulated.xe2x80x9d So, for example, a researcher can compare a normal colon cell with a malignant colon cell and thereby determine which genes are being expressed or not expressed in the aberrant cell. The regulatory cites of genes serves as key targets for drug therapy.
Proper performance of a DNA array depends on two basic factors: 1) retention of the immobilized probe nucleic sequences on the substrate, and 2) hybridization of the target sequence to the immobilized probe sequence, as measured by fluorescence emission from the tagged target sequence. The DNA probe material must be retained on the surface of the substrate through a series of washing, blocking, hybridizing, and rinsing operations that are commonplace in DNA hybridization assays. An excessive loss of probe DNA sequences can lead to a low fluorescent-signal-to noise ratio and uncertain or erroneous results.
DNA arrays have for years been printed onto organic, micro-porous membranes such as nylon or nitrocellulose. The densities at which one can print DNA solutions onto these types of organic micro-porous membranes is limited because of the tendency for the DNA solution to wick laterally through the membrane, thus causing cross-talk and contamination between adjacent locations. Others have employed a flat, non-porous substrate surface made from glass. (See for example, U.S. Pat. No. 5,744,305, incorporated herein by reference.) These substrates, however, have also been found wanting, since they do not retain the probe molecules as well as porous substrates.
The present invention proposes to use a substantially flat, porous, inorganic substrate surface to enhance retention of nucleic moieties for high-density arrays. The porous surface provides increased surface area for immobilizing DNA probe molecules, which increases the density of DNA binding sites per unit cross-sectional area of the substrate. The increased number of possible binding sites per unit area results in greater retention of immobilized DNA probes and the emission of an increased signal when hybridized with target molecules. A porous inorganic surface that is properly treated with a coating of a binding agent, such as a cationic polymer, can also prevent lateral cross-talk. Moreover, the present invention can both enhance sensitivity and improve threshold detection of fluorescence markers.
The present invention relates to a device for performing multiple biological or chemical assays. The device includes a substantially planar substrate for attaching a high-density array of biological or chemical analytes. The substrate comprises a porous, predominantly inorganic layer adhered to a flat, rigid, non-porous, inorganic understructure, preferably having a coefficient of thermal expansion (CTE) compatible with that of the porous inorganic layer (CTExc2x115xe2x88x9225%). Preferably the CTEs are matched. The porous inorganic layer is characterized as having a plurality of interconnected voids of a predetermined mean size of not less than about 0.1 xcexcm dispersed therethrough, and having void channels that extend through to a top surface of the porous inorganic layer. The voids are defined by a network of either contiguous or continuous inorganic material having a predetermined mean size of not less than about 0.1 xcexcm, and the inorganic material and contents of the voids exhibit a high contrast in their indices of refraction relative to each other.
The substrate further comprises a uniform coating of a binding agent over at least a part of the surface area of the voids and the top surface of the porous inorganic layer, and preferably an interlayer disposed between the porous inorganic layer and the inorganic understructure. The interlayer having a coefficient-of-thermal-expansion compatible with said porous inorganic layer and said inorganic understructure.
The inorganic material is characterized as a material that is non-absorbing and transparent to light when in the form of a solid of an amorphous or single crystal material, such as a glass or a metal oxide. More particularly for example, the material is, a silicate, aluminosilicate, boroaluminosilicate, or borosilicate glass, or TiO2, SiO2, Al2O3, Cr2O3, CU/O, ZnO, or ZrO2 layer.
The porous inorganic layer of the substrate has a thickness of at least about 5 xcexcm. The network of inorganic material is formed by adhesion or sintering of the inorganic material particles to each other. The particles have a predetermined mean size preferably in the range of about 0.5 xcexcm to about 5 xcexcm, more preferably in the range of about 0.5 xcexcm to about 3.5 xcexcm. The voids within the porous inorganic layer have a predetermined mean size preferably in the range of about 0.5 xcexcm to about 5 xcexcm, and also, more preferably in the range of about 0.5 xcexcm to about 3.5 xcexcm. And, the content of the voids consists of either a gas, a liquid, or a solid.
The invention also relates to a method of making the substrate used in the device. The method comprises the following steps: providing a flat, rigid, non-porous, inorganic understructure, applying a porous inorganic layer having a coefficient of thermal expansion compatible with that of the inorganic understructure to a top surface of the inorganic understructure. The porous inorganic layer is formed by a process that comprises: applying a layer of individual particles of an inorganic material to a top surface of the inorganic understructure, the particles having a predetermined mean size of not less than about 0.1 xcexcm, and forming a network of either contiguous or continuous inorganic material from the individual particles to create a plurality of interconnected voids of a predetermined mean size of not less than about 0.1 xcexcm dispersed through-out the porous inorganic layer, and having void channels that extend through to a top surface of the porous inorganic layer. Then, the inorganic material and contents of the voids are configured to exhibit a high contrast in their indices of refraction relative to each other; and the top surface and internal surfaces of the porous inorganic layer is prepared for binding biological or chemical analytes. To prevent emission of a high background signal due to non-specific binding of target DNA molecules after hybridization, the substrate can be pretreated with blocker agents or deactivators after the probe DNA has been applied.
The method may further comprise applying an interlayer disposed between the inorganic understructure and the porous inorganic layer. The interlayer has a coefficient-of-thermal-expansion compatible said inorganic understructure and with said porous inorganic layer. A tape casting or a screen printing process may be used in the forming of the network of continuous inorganic material from the individual particles.